Evidence suggests that the c-Jun protooncogene delta (δ) domain (amino acids 31-60) helps regulate the transcriptional activating capacity of c-Jun by modulating the amino-terminal phosphorylation of this protein. By using a peptide encoding the δ domain and purified amino-terminal c-Jun protein kinase, we demonstrate that the δ domain peptide inhibits phosphorylation of the amino terminus of both c-Jun and the related protein JunD. The δ domain peptide inhibited the activation of the c-Jun amino-terminal protein kinase by phorbol esters in permeabilized U937 leukemic cells. Mutation of c-Jun followed by transfection into U937 leukemic cells demonstrated that partial deletions of the δ domain are sufficient to block phosphorylation of the amino terminus of c-Jun. In vitro deletion of the amino-terminal (amino acids 31-44) half of the δ domain inhibited the phosphorylation of c-Jun. However, deletion of the carboxyl-terminal (amino acids 45-60) half only partially inhibited c-Jun phosphorylation. Therefore, these results indicate that the δ domain sequence is an important regulator of c-Jun amino-terminal phosphorylation.